By definition, the existence of periplasmic proteins requires some mechanism for the translocation of these proteins from the cytoplasm where they are synthesized through the cytoplasmic membrane to the periplasmic space. Often, when the bacterial cell synthesizes one of these extracytoplasmic proteins, the final form of the protein (the "mature protein") is synthesized with a peptide presequence (the "leader sequence") linked by a peptide bond to the amino terminus of the mature protein. The protein molecule with the leader sequence attached to the mature protein is called the "precursor protein". This leader sequence is essential to the transport of many extracytoplasmio proteins across membrane surfaces and is cleaved from the molecule at some point during the transport process, thus releasing the mature protein. In E. coli, for example, periplasmic proteins found to contain such leader sequences include .beta.-lactamase, alkaline phosphatase as well as a peptidase and a protease.
The leader sequences of many E. coli extracytoplasmic proteins have been analyzed and a common structure has emerged. The amino terminal ends of the leader sequences are basic in nature and are followed by a highly hydrophobic region. From this and other research two models have been proposed to explain how the sequences may function in the transport of proteins across membrane surfaces.
According to the signal hypothesis of Blobel and Dobberstein [J. Cell Biol. 67: 835-851 (1975)] the hydrophobic region of the leader sequence of a nascent protein molecule acts to bind the protein-ribosome complex to the cytoplasmic membrane during formation of the protein. In binding to the membrane, the leader sequence interacts with a membrane receptor protein which is then activated to form a hydrophilic channel through the membrane into which the growing protein chain is inserted and through which it is translocated to the periplasm. At some point during this transport, the leader sequence is removed from the protein molecule and the mature protein appears in the periplasm.
The loop model of Inouye and Halegoua, [CRC Crit. Rev. Biochem 7: 339-371 (1980)] proposes that, as the extracytoplasmic protein is translated, the basic amino terminal region of the leader sequence binds onto the membrane, its basic amino acid residues interacting with the negatively charged membrane surface. The hydrophobic region of the sequence is then inserted into the membrane forming a loop and interacting with the lipid that comprises a large part of the membrane. As translation proceeds, the looped leader sequence traverses the membrane and is cleaved, leaving a track through which the nascent protein can move (as it is formed) through the membrane to the periplasm.
In recent years, researchers have attempted to utilize the translation and transport mechanisms in E. coli to produce and obtain proteins not normally produced by the microorganism, including proteins of other bacteria and proteins of viruses and higher organisms. These proteins not native to E. coli are called "foreign" proteins. One technique involves insertion of a DNA sequence coding for a ""foreign" protein of interest (e.g., a eukaryotic protein) at some point after the leader sequence (i.e., distal to the carboxy terminus of the leader sequence) of a bacterial gene coding for the precursor form of an extracytoplasmic protein. The protein resulting from such a constructed gene is called a "fusion protein" consisting of a foreign protein usually attached to a bacterial protein portion. (After this gene has been constructed it is usually introduced into the bacterium by transformation with a compatible and replicable plasmid vector. Alternatively, the constructed gene can be inserted into the bacterial chromosome.) When the constructed gene is transcribed and as the protein of interest is formed it is transported to the periplasm via the bacterial transport mechanism discussed above. Recently, for example, the rat pre-proinsulin gene was inserted by recombinant DNA techniques into the E. coli .beta.-lactamase gene [Villa-Komaroff, et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731 (1978 )]. .beta.-lactamase is a periplasmic enzyme which, in its precursor form, carries a twenty-three amino acid leader sequence. The fusion protein resulting from the expression of the fused .beta.-lactamase and pre-proinsulin genes was transported to the periplasm via the above-described normal transport mechanism.
European patent application No. 0,006,694, filed Apr. 6, 1979, listing Gilbert, et al. as inventors, discloses the production of such genetically-engineered fusion proteins by expression of DNA sequences having the gene coding for a "foreign" protein of interest attached to a DNA sequence coding for a leader sequence of a periplasmic protein. This procedure, however, merely provides a way to transport the protein of interest to the periplasmic space. The protein must still be extracted and isolated from this compartment. Gilbert, et al. also briefly mention attachment of the gene of interest to the leader sequence of a secretory, or extracellular, protein. In theory, in such an arrangement the protein of interest would be excreted beyond the periplasm into the fermentation medium. However, there are no known E. coli proteins which are externalized, that is, transported beyond the outer membrane.
U.S. Pat. No. 4,336,336, filed Jan. 12, 1979, listing Silhavy, et al. as inventors, discloses a method of producing fusion proteins which are transported into the outer membrane. This method provides for the fusion of a gene coding for a cytoplasmic bacterial protein with the gene for a non-cytoplasmic carrier protein thereby producing a fusion protein which is carried to the outer membrane. In addition, it is asserted that the method could be used to insert a "foreign" gene (e.g., coding for a eukaryotic protein) into the already constructed fusion gene.
However, as with the Gilbert, et al. invention discussed above, this method does not result in transportation of the protein of interest to the extracellular medium. Conventional extraction methods must still be employed to isolate the protein.
The conventional isolation of bacterial protein from the periplasm of gram-negative bacteria poses problems owing to the tough, rigid cell walls that surround these cells. The bacterial cell wall maintains the shape of the cell and protects the cytoplasm from osmotic pressures that may cause cell lysis; it performs these functions as a result of a highly cross-linked peptidoglycan (also known as murein) backbone which gives the wall its characteristic rigidity.
E. coli and the other gram-negative bacteria have a complex cell membrane structure. There is an outermost membrane exterior to the peptidogylcan layer of the cell wall. Interior to the peptidoglycan layer lies the cytoplasmic membrane. Between the outer and cytoplasmic membranes is an aqueous compartment, called the periplasm or periplasmic space, where certain enzymes and other proteins are located. Since these periplasmic proteins are located outside the cytoplasmic membrane they can be isolated without disrupting the cytoplasmic membrane and thus can be obtained relatively free from cytoplasmic contaminants. The selective extraction of these periplasmic proteins without release of cytoplasmic proteins, however, requires disruption of the cell wall, either by mechanical or chemical means while leaving the inner, or cytoplasmic, membrane intact. Thus, some means for removing the cell wall or increasing its permeability is necessary for isolation of the periplasmic proteins free from cytoplasmic contaminants.
A widely used technique of cell wall removal is enzymatic treatment of the cell culture with lysozyme which hydrolyzes the peptidoglycan backbone of the cell wall. The method was first developed by Zinder and Arndt [Proc. Natl. Acad. Sci. USA 42: 586-590 (1956)] who treated E. coli with egg albumin (which contains lysozyme) to produce rounded cellular spheres later known as spheroplasts. These structures retained some cell wall components but had large surface areas in which the cytoplasmic membrane was exposed. However, there are several disadvantages to use of the lysozyme method for isolating periplasmic proteins. Firstly, the cells must be treated with EDTA (ethylenediamine tetraacetic acid) or high pH, both of which tend to weaken them. Secondly, the method is not suitable for lysis of large amounts of cells because the enzyme is inefficient and there is difficulty in dispersing the enzyme throughout a large pellet of cells. Thirdly, since treatment with lysozyme results in the formation of spheroplasts, the treatment must be performed in an osmotic stabilizing solution to prevent lysis. In addition, attempts to culture spheroplasts indefinitely, in order to harvest protein excreted beyond the cytoplasmic membrane, would encounter problems of spheroplast stability.
Another method of extracting periplasmic proteins does not involve physical removal of the cell wall to expose the periplasm but rather entails subjecting the cells to a severe osmotic shock which causes the release of certain periplasmic proteins. The cells are placed in a hypertonic sucrose medium containing EDTA which causes them to lose water and shrink, the cytoplasmic membrane drawing away from the cell wall. The cells are then placed in a magnesium chloride "shock" solution which drastically decreases the osmotic pressure outside the cell causing water to rush into the cell which swells the cell and seems to propel the periplasmic proteins to the exterior of the outer membrane. It should be noted that while EDTA increases permeability of the membrane surface in E. coli, it will not by itself cause release of periplasmic enzymes; rather, both osmotic shock and EDTA treatment are necessary. Moreover, it would be extremely difficult to shock large amounts of cells.
Many other methods which totally disrupt cells are non-selective for periplasmic proteins.
Lastly, E. coli mutants that leak various periplasmic enzymes have been isolated. For example, Lopes, et al. [J. Bacteriol. 109(2): 520-525 (1972)] treated E. coli cells with a mutagen such as nitrosoguanidine and mutants excreting periplasmic enzymes were selected by enzyme assay systems. Such mutants included those leaking ribonuclease I, endonuclease I and alkaline phosphatase. It is believed that these mutants are deficient in some component of the outer bacterial membrane leading to an increase in the cells' permeability. In addition, several excreted periplasmic proteins have been separated from the culture medium by antibody precipitation or SDS-polyacrylamide gel electrophoresis in order to characterize these "periplasmic leaky" mutants [see, for example, Anderson, et al., J. Bacteriol. 140(2): 351-358(1979) and Lazzaroni and Portalier, J. Bacteriol. 145(3): 1351-1358 (1981)].